Epoxy fatty acids contain oxygen bridges across adjacent carbon atoms at a single or multiples positions in the acyl chain, making them highly reactive and prone to cross-linking. These properties make epoxy fatty acids valuable raw materials, particularly for epoxy coating and plasticizers. Currently, epoxidized soybean and linseed oils are produced by introducing an epoxy group across the double bond of polyunsaturated fatty acids. This is a costly process and it would likely be more economical if the biosynthetic reactions in oilseed themselves converted the polyunsaturated fatty acids into epoxy fatty acids. However, there currently is no known way to produce a commercial oilseed that accumulates epoxy fatty acids by conventional breeding and genetics.
Certain genotypes of several plant species accumulate high level of epoxy fatty acids in the seed oil. Epoxy fatty acids, like vernolic (E-12,13-epoxyoctadeca-E-9-enoic) and coronaric (E-9,10-epoxyoctadeca-E-12-enoic) acids, have been found as a component of the seed oil of species represented by a number of plant families such as Asteraceae, Euphorbiceae, pnagraceae, and Valerianaceae (Smith, 1970). One of the highest known accumulators of vernolic acid is Vernonia galamensis in which vernolic acid constitues 80% of triglyceride fatty acids (Perdue, 1989; Pascal and Correal, 1992; Thompson et al., 1994). Stokesia laevis is in the Asteraceae family, and the seed soil is also made up of about 60-70% vernolic acid (Gunstone, 1993).
Many plants are known to possess enzymes that transform unsaturated fatty acids into epoxy fatty acids (Gardner, 1991; Gardner et al., 1991; Blee, 1998). The process by which the seeds of certain species of Vernonia, Stokesia and Euphorbia appears to be due to an enzyme not present in major commercial oilseeds. Biochemical studies by Bafor et al. (1993) indicate that developing seeds of these plants contain an enzyme known as epoxygenase which converts linoleic acid into vernolic acid in a one step reaction. Although many plants including soybeans have enzymes such as lipoxygenase and peroxygenase that can produce epoxy fatty acids in some disease resistance reactions (Blee, 1998), they do not have mechanisms for accumulation of epoxy-triglycerides in their seeds unlike epoxy triglyceride accumulators such as Stokesia mentioned above (Bafor et al., 1993; Hildebrand et al., 2001).
The original studies on epoxy fatty acid synthesis and accumulation in oilseeds by Bafor et al. (1993) indicated that the epoxy fatty acid that accumulated in seeds of E. lagascae, vernolic acid, is synthesized by an epoxygenase enzyme which is a P450 monooxygenase enzyme. It was assumed that other epoxy fatty acid accumulators such as Vernonia similarly synthesized epoxy fatty acids by P450 monooxygenase enzymes.
However studies by Seither (1996), Seither, Avdiushko et al. (1996, 1997) indicated that delta12 fatty acid desaturase-like enzymes are responsible for vernolic acid biosynthesis in epoxy fatty acid accumulators of the Asteraceae such as Vernonia, Crepis and Stokesia unlike the Euphobiacea epoxide accumulator, E. lagascae, which relied on P450 monooxygenase (Seither, 1996, Seither, Avdiushko et al., 1996, Seither, Avdiushko et al., 1997).
A seed specific P450 monooxygenase family enzyme was cloned from Euphorbia lagascae. The international patent application WOO2/08269 deals with the description of an enzyme of the cytochrome P450 mono-oxygenase class that is responsible for the epoxide formation. It is encoded by a nucleic acid isolated from Euphorba lagascae developing seed cDNA library. This patent application also discloses the nucleotide and amino acid sequence of the cytochrome P450 mono-oxygenase of Euphorba lagascae. The fatty acid compositions were monitored, showing accumulation of epoxy fatty acids in transformed plants.
Genes from Vernonia galamensis and Crepis palaestina have been isolated, and when expressed in plants or yeast, the encoded proteins are capable of converting linoleic acid to vernolic acid (U.S. Pat. No. 5,846,784, WO 98/56922, U.S. Pat. No. 6,329,518 and WO 98/46762).
U.S. Pat. No. 5,846,784 relates to a nucleic acid encoding Vernonia galamensis fatty acid modifying enzymes. This nucleic acid is used to transform microbial and soybean embryogenic cells. The expression of this nucleic acid results in production of altered levels of a Vernonia galamensis fatty acid modifying enzyme in the transformed cells.
WO 98/56922 international patent application claims the priority of the above cited U.S. patent, it contains further example dealing with the transformation of corn cells and Arabidopsis cells. The expression of the Vernonia fatty acid epoxidase has been monitored in Arabidopsis seeds. The putative vernolic acid represented about 1.5% of the total fatty acids.
U.S. Pat. No. 6,329,518 and international patent application WO98/46762 describe the isolation and characterization of an isolated nucleic acid molecule which encodes a fatty acid epoxygenase from Crespis palaestina. They also describe a genetic construct comprising said nucleic acid molecule operatively linked to a promoter sequence that is capable to be transcribed. The protein encoded by this nucleic acid molecule is a delta12 epoxygenase polypeptide which catalyses the epoxygenation of a carbon double bound in linoleic acid to form vernolic acid.
However, the epoxygenase activities from these last plants producing vernolic acid have been shown to be different from the E. lagascae enzyme. Unlike the E. lagascae enzyme, they are related to the endoplasmic reticulum-localized fatty acid desaturases (WO 94/11516). These fatty acid epoxygenase enzymes are related in sequence to the class of membrane bound enzymes responsible for fatty acid desaturation and fatty acid hydroxylation (Broun and Somerville, 1997). Therefore, there are two distinct classes of genes encoding enzymes capable of vernolic acid epoxide group formation, one that is cytochrome P450-dependant, and the other that is related to the fatty acid desaturases and hydrolases.
The epoxygenase responsible for vernolic acid production in Stokesia laevis developing seed is a novel protein.